GSM8046084: Optix_library_2; Drosophila melanogaster; RNA-Seq
Whole brains were extracted in ice-cold Schneider's media. The brains were then transferred into DPBS, we used an epifluorescence microscope to remove brains with ectopic mOPC GFP expression, and placed the brains back in Schneider's media. The brains were then incubated at 25˚C in Schneider's media with 2mg/mL Collagenase and 2mg/mL Dispase, for 1hr 30min (adult brains) or for 30min (pupal brains), then washed 3 times with ice-cold Schneider's media to stop the dissociation. Optic lobes were then separated from the central brain, transferred in ice-cold DPBS + 0.04% BSA, and washed twice with ice-cold DPBS + 0.04% BSA. For pupal brains, the separation between central brain and optic lobes can be seen as a difference in texture: the optic lobe forms a smoother and cohesive structure that can be removed using a fine tungsten needle. Optic lobes and 150μL of DPBS + 0.04% BSA were then transferred to a low-bind Eppendorf, and dissociated by pipetting vigorously 50 times, resting the cells for 1 minute on ice, and pipetting 50 times more. The cell suspension was then checked under a dissecting microscope, and we pipetted up to 50 more times if the size of remaining clumps was too significant. We did not try to dissociate small clumps since they are most likely neuropils, not soma, and indeed we empirically noticed a better quality of the data obtained after lowering pipetting repetitions. The suspension was then passed through a 20µm mini-PluriStrainer, and the filter was washed with 400μL of DPBS + 0.04% BSA which we also collected (pipetting from the opposite side may be necessary to collect the entire cell suspension). Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index )